Staphylococcus is a leading causative agent for hospital-acquired infections. S. aureus can cause skin, bone, heart valve, blood, and lung infection resulting in severe diseases such as endocarditis, osteomyelitis and pneumonia, while S. epidermidis mainly causes foreign body infections. Biofilm formation has long been recognized as the major virulence factor for S. epidermidis and has been recently implicated to play a key role in endocarditis, osteomyelitis and indwelling device associated infections caused by S. aureus. Biofilm, a community of microorganisms attached to a solid surface, is generally highly resistant to antibiotics and host immune defense mechanisms and therefore contributes to persistence of diseases. The mechanism of biofilm formation in staphycococci is largely unknown and much remains to be explored. Recently, we have identified and initially characterized a novel regulator, Rbf, which positively regulates biofilm formation in S. aureus. We demonstrated that Rbf affected biofilm formation in various strains and it affected many target genes. We also showed that rbf was regulated by two regulators, agr and sigB, and was an important virulence factor in promoting bacterial persistence in a mouse foreign body infection model. In this application we propose to continue our studies by accomplishing four specific aims: (1) to study the molecular architecture of the rbf locus and to analyze rbf expression in the murine model of foreign body infection;(2) to identify the rbf target genes by proteomic and microarray approaches and to study the mechanism of target gene regulation by rbf;(3) to study regulation of rbf by known regulators, agr and sigB, and to identify additional genes affect Rbf expression;(4) to investigate the role of Rbf in native tissue infection using a biofilm relevant model of septic arthritis in mice. Staphylococcal infections have become more difficult to treat due to the emergence of antibiotic resistance strains. Moreover, bacteria that form biofilm will be protected in the biofilm from treatments even with effective antibiotics. Understanding biofilm formation and its regulation at the molecular level will contribute to new knowledge on the mechanisms of pathogenesis of biofilm-associated infecitons, which will lead to the development of novel therapeutic strategies for treating biofilm-related Staphylococcal diseases.